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Research ArticleArticle

First-Pass Midazolam Metabolism Catalyzed by 1α,25-Dihydroxy Vitamin D3-Modified Caco-2 Cell Monolayers

Jeannine M. Fisher, Steven A. Wrighton, Paul B. Watkins, Phyllissa Schmiedlin-Ren, Justina C. Calamia, Danny D. Shen, Kent L. Kunze and Kenneth E. Thummel
Journal of Pharmacology and Experimental Therapeutics May 1999, 289 (2) 1134-1142;

Abstract

Cytochrome P-450 (CYP) 3A4 accounts for approximately 50% of all P-450s found in the small intestine (Paine et al., 1997) and contributes to the extensive and variable first-pass extraction of drugs such as cyclosporine and saquinavir. We recently demonstrated that CYP3A4 expression in a differentiated Caco-2 subclone is increased when cell monolayers are treated with 1α,25-dihydroxy-vitamin-D3 (Schmiedlin-Ren et al., 1997). This improved metabolic capacity permits the in vitro modeling of first-pass intestinal metabolic kinetics. Midazolam (MDZ) 1′-hydroxylation was used as a specific probe for CYP3A-mediated metabolism in modified Caco-2 monolayers. Caco-2 cells were grown to confluence on laminin-coated culture inserts, and then for two additional weeks in the presence of 1α,25-dihydroxy vitamin-D3. Cell monolayers were subsequently exposed to MDZ for varying lengths of time and concentrations. The amount of MDZ in the monolayer increased rapidly after apical drug administration, reaching a pseudo steady state within 6 min. The cellular uptake rate was considerably slower after a basolateral dose. By either route of administration, the rate of 1′-hydroxymidazolam formation was stable and linear for 2 h. Under basolateral sink conditions and low apical MDZ dosing concentration (1–8 μM), the first-pass extraction ratio was found to be ∼15%. Higher dosing concentrations led to saturation of the hydroxylation reaction and reduction in the extraction ratio. The modified Caco-2 cell monolayer is an excellent model for studying drug absorption and first-pass intestinal metabolic kinetic processes. In this system, the selective CYP3A probe MDZ was rapidly absorbed, yet extensively metabolized, as is observed in vivo.

Cytochrome P-450s (CYP) are one of the most important classes of drug-metabolizing enzymes found in humans and are responsible for the elimination of a wide variety of xenobiotics (Guengerich, 1995). Although these enzymes are primarily concentrated in the liver, several extrahepatic tissues including the human intestine are also known to express appreciable levels of this class of enzymes. CYP3A4 is the most abundant of all P-450 isoforms in the liver and small intestine (Watkins et al., 1987;de Waziers et al., 1990; Paine et al., 1997). Immunoblot analysis indicates that the median microsomal CYP3A protein content (per mg total protein) in the duodenal mucosa is approximately one-half that found in the liver (de Waziers et al., 1990; Paine et al., 1997).

The presence of this major oxidizing enzyme in the small intestine, specifically at the tips of the mucosal microvilli (Kolars et al., 1994), makes it a potentially important site for first-pass drug metabolism after oral administration. For example, expression of CYP3A4 within villous epithelial cells is thought to result in significant first-pass metabolism of many clinically important substrates (Thummel et al., 1997), including cyclosporine, saquinavir, midazolam (MDZ), nifedipine, verapamil, and tacrolimus, thereby contributing to their low overall oral bioavailability. In addition, clinically relevant drug-drug interactions can result from inhibition or induction of intestinal CYP3A, as seen with cyclosporine and ketoconazole (Gomez et al., 1995) and with verapamil and rifampin (Fromm et al., 1996). Despite a growing recognition of the potential effect of intestinal CYP3A on oral drug bioavailability and efficacy, a clear understanding of the kinetic factors governing the extent of intestinal first-pass metabolism in the absence and presence of enzyme modifying agents is lacking.

Caco-2 cells are used widely in pharmaceutical research as an in vitro model for studying intestinal drug absorption and transport processes (Meunier et al., 1995; Boulenc, 1997). This colon-carcinoma derived cell line is easily grown on a solid, permeable membrane to achieve a confluent monolayer of fully differentiated cells with apically directed microvilli and tight occluding junctions. In addition to the relevant structural features that make Caco-2 cells well suited for drug absorption studies, the differentiated cells are also known to produce important transport proteins such as P-glycoprotein (P-gp;Hunter et al., 1993) and some xenobiotic-metabolizing enzymes including CYP1A1, glutathione-S-transferase, and phenol-sulfotransferase (Meunier et al., 1995; Boulenc, 1997). Although CYP3A4 is abundant in the epithelium of a normal human small intestine, standard culturing conditions with Caco-2 cells do not lead to significant expression of the enzyme. There is evidence of CYP3A5 expression and activity in Caco-2 cells and a subclone TC7 (Gan et al., 1996; Raeissi et al., 1997); however, CYP3A4 and CYP3A5 can produce different product ratios from the same substrates (Wrighton et al., 1990; Gorski et al., 1994). Therefore, by both quantitative and qualitative criteria, standard Caco-2 cell monolayer cultures are not a suitable model for first-pass metabolism.

In an effort to rectify this deficiency, Crespi et al., (1996) recently reported the transfection of Caco-2 cells with an extrachromosomal vector containing the CYP3A4 gene. Microsomes prepared from these transfected cells contain approximately 45 pmol P-450/mg protein. The intact cells showed significant catalytic activity, as measured by testosterone 6β-hydroxylation. However, multiple passages of these Caco-2 cells lead to loss of the plasmid and CYP3A4 expression.

Recent work in our laboratories has shown that CYP3A4 expression can be up-regulated in Caco-2 cells with the use of 1α,25-dihydroxy vitamin-D3 (1α,25-(OH)2-D3), a naturally occurring hormone. CYP3A4 expression in a Caco-2 subclone was increased when cells were grown to confluence on an extracellular matrix such as Matrigel (Collaborative Biomedical Products, Bedford, MA) or laminin and then treated with 0.25 μM 1α,25-(OH)2-D3 for 2 to 3 weeks (Schmiedlin-Ren et al., 1997). In addition to the approximately 3-fold increase in CYP3A4 apoprotein, there was also a 50-fold increase in its activity as measured by midazolam 1′-hydroxylation.

Our previous experiments with the modified Caco-2 subclone focused on MDZ incubation times up to 24 h in the presence of apical and basolateral medium containing fetal bovine serum (FBS). The objective of the present work was to characterize MDZ first-pass metabolic extraction and metabolite distribution across 1α,25-(OH)2-D3-treated Caco-2 monolayers under conditions of first-order product formation in the absence of extracellular serum proteins.

Experimental Procedures

Materials.

Caco-2 cells (American Type Culture Collection HTB37) were cloned by limiting dilution as described previously (Schmiedlin-Ren et al., 1997). Dulbelcco’s modified Eagle’s medium (DMEM), non-essential amino acids (NEAA), penicillin, streptomycin, and Hanks’ balanced salt solution were obtained from GIBCO (Grand Island, NY). FBS was purchased from Hy-Clone Laboratories, Inc. (Logan, UT). Uncoated and commercially coated track-etched polyethylene terephthalate (PET) inserts and mouse laminin were obtained from Collaborative Biomedical Products (Bedford, MA). 1α,25-(OH)2-D3 was obtained from Calbiochem Corp. (La Jolla, CA).N-Methyl-N-(t-butyl-dimethylsilyl)trifluoroacetamide (MTBSTFA) was purchased from Pierce Chemical Co. (Rockford, IL). MDZ, 15N3-MDZ, 1′-hydroxymidazolam (1′-OH MDZ), 4-OH MDZ, and 1′-[2H2]1′-OH MDZ were gifts from Roche Laboratories (Nutley, NJ). Acetonitrile and ethyl acetate were purchased from Fisher Scientific Co. (Santa Clara, CA). SDS-polyacrylamide gel electrophoresis reagents (SDS, acrylamide, ammonium persulfate, andN,N,N′,N′-tetra-methyl-ethylenediamine) were purchased from Bio-Rad (Hercules, CA). Nitrocellulose was purchased from Schleicher & Schuell (Keene, NH). 5-Bromo-4-chloro-3-indoyl phosphate and nitroblue tetrazolium was purchased from Kirkegaard and Perry (Gaithersburg, MD). Anti-rabbit IgG alkaline phosphatase conjugate, EDTA, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). All other chemicals were of tissue culture grade. Stock solutions of MDZ and 1α,25-(OH)2-D3 were prepared in DMSO and absolute ethanol, respectively.

Cell Culture Conditions.

The Caco-2 subclone, P27.7 (Schmiedlin-Ren et al., 1997), was obtained at passage 12 and grown on culture dishes in passage medium consisting of DMEM containing 25 mM glucose and 4 mM l-glutamine, 0.1 mM NEAA, 100 U/ml sodium penicillin G, 100 μg/ml streptomycin, and 20% heat-inactivated FBS. All experiments were performed with cells at passage number 19 or 20. Unless otherwise noted, cells were seeded onto laminin-coated PET inserts at 5.2 × 106 cells/cm2 and grown in complete growth medium (passage medium supplemented with 45 nMdl-α-tocopherol) until confluent. Upon achieving confluence, cell monolayers were fed for 2 weeks every 2 to 3 days with complete differentiation medium (DM) containing DMEM, 0.1 mM NEAA, 100 U/ml sodium penicillin, 100 μg/ml streptomycin, 0.1 μM sodium selenite, 3 μM zinc sulfate, 45 nM dl-α-tocopherol, 0.25 μM 1α,25-(OH)2-D3, and 5% heat-inactivated FBS.

On a given experiment day, transepithelial electrical resistance (TEER) readings were recorded before initiation of the experiment. DM was removed and cells were washed three times with 1 ml of DMEM before addition of experimental medium. Modified DM (absent 1α,25-(OH)2-D3 and FBS) was spiked with midazolam to the desired concentration and added to the appropriate culture insert compartment (1.5 ml volume for apical and basolateral compartments). The final concentration of DMSO in the dosing medium was always <1%. Apical and basolateral media were collected upon completion of the indicated incubation period and frozen at −20°C pending analysis. Cells were quickly rinsed with 1 ml of DMEM and scraped into a fresh 1 ml of medium and immediately frozen at −20°C.

Except for the results described in the sections Concentration Dependence of 1′-OH and 4-OH MDZ Formation, Intraday Variability in 1′-OH MDZ Formation and Distribution, and Saturable First-Pass Metabolic Extraction, all sets of data points (grouped by collection time) represent results from a single culture.

Monolayer Integrity.

TEER was the primary determinant of cell monolayer integrity. Resistance (ohm) was measured on each culture immediately before an experiment with a Millipore Millicell electrical resistance system (Bedford, MA). For a given set of cell cultures, a single insert that did not contain cells was measured for background resistance. TEER was determined as the product of the background-corrected resistance and the surface area of the insert (4.2 cm2).

Extracellular Matrix Proteins.

In specified experiments, cells were seeded onto commercially available culture inserts precoated with 25 μg/cm2 mouse laminin. In all other experiments, control inserts were hand-coated with 5 μg/cm2 mouse laminin according to Collaborative Biomedical Product’s specification immediately before seeding cells. Briefly, mouse laminin was thawed at 4°C overnight and diluted into DMEM to a final concentration of 21 μg/ml. Each 4.2 cm2 PET insert was coated with 1 ml of the diluted laminin and incubated at room temperature for 1 h. The medium was then aspirated and the coated inserts were rinsed three times with 1 ml of fresh DMEM and then immediately seeded with Caco-2 cells. Cells grown on hand-coated laminin inserts were morphologically identical with those grown on commercially coated inserts, as determined by electron microscopy, although the laminin layer appeared thinner (data not shown). Additionally, cells achieved confluence after approximately 4 days on hand-coated inserts compared with 7 days with commercially coated inserts.

Preparation of Caco-2 Cell Fractions.

Caco-2 cells were grown on 28 commercially coated laminin inserts and treated with 1α,25-(OH)2-D3 for 2 weeks postconfluence. After recording the TEER on each culture, cell monolayers were rinsed three times with 1 ml DMEM, scraped with a metal spatula into 500 μl of homogenizing buffer (100 mM potassium phosphate, 0.25 M sucrose, and 1 mM EDTA, pH 7.4), and collected in a 50-ml conical tube. Each insert was then rinsed with an additional 500 μl of homogenizing buffer, which was transferred to the same tube. The cell homogenate was subjected to centrifugation at ∼300g at 4°C for 10 min, at which time the supernatant was aspirated and the pellet was reconstituted in approximately 6 ml of homogenizing buffer. Cells were then transferred to a 10-ml glass Wheaton tube and hand-homogenized (20 strokes) and drill-homogenized (five strokes) on ice with a Teflon-tipped pestle. A 300 μl aliquot was immediately frozen for protein and immunochemical analysis, and the remaining homogenate was subjected to a low-speed centrifugation at 600g for 5 min, then at 21,400g for 25 min at 4°C. The resulting yellow pellet (1st pellet) was resuspended in 1 ml of homogenizing buffer and frozen before Western blot analysis. A 500 μl aliquot of the supernatant was immediately frozen, whereas the remainder was diluted to 60 ml and centrifuged at 110,000g for 70 min at 4°C. The resulting microsomal pellet (2nd pellet) was resuspended in 500 μl of wash buffer (10 mM potassium phosphate, 1 mM EDTA, pH 7.4) and centrifuged at 110,000g for 70 min at 4°C. The final pellet was reconstituted in 250 μl of storage buffer (100 mM potassium phosphate and 20% glycerol, pH 7.4) and immediately frozen with the other fractions at −80°C. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin as the reference standard.

Western Blot Analysis of CYP3A.

Total CYP3A protein in modified Caco-2 cell fractions was immunoquantified and compared to human liver and duodenal microsomal CYP3A content. Liver and small intestinal microsomes were prepared as described previously (Paine et al., 1997). After dilution in buffer (final concentrations: 60 mM Tris-HCL, pH 7.4; 20% glycerol; 0.2% Emulgen 911; 2% SDS; 5% β-mercaptoethanol), samples were boiled for 3 min and loaded onto a 9% acrylamide/0.1% SDS gel. Protein components of Caco-2 homogenate (50 μg), 1st pellet (50 μg), 21,400g supernatant (40 μg), and final 110,000g pellet (20 μg), and human intestinal (20 μg) and liver (10 μg) microsomes were separated by electrophoresis (Paine et al., 1997). Purified expressed human CYP3A4 and CYP3A5 standards (0.5 and 1 pmol each) were prepared in the same manner and loaded onto the same gel into lanes parallel to cellular fractions. Transfer of proteins to nitrocellulose sheets, and CYP3A immunoblot detection and quantification were described previously (Paine et al., 1997).

MDZ, 1′-OH MDZ, and 4-OH MDZ Assays.

Formation of 1′-OH MDZ was the primary measure of MDZ metabolism by Caco-2 cell cultures at low (<3 μM) dosing concentrations (Schmiedlin-Ren et al., 1997). Because 4-OH MDZ formation is increasingly favored at higher concentrations in microsomal incubations (Gorski et al., 1994; Paine et al., 1997), both the 1′-OH and 4-OH metabolites were measured in Caco-2 cultures incubated with initial apical MDZ concentrations greater than 3 μM to assess saturable metabolite kinetics. 4-OH MDZ was not quantitated in all samples to conserve a limited quantity of15N3-MDZ-labeled 4-OH MDZ internal standard.

Measurement of MDZ levels in the apical and basolateral medium and cell mass were used to calculate the extent of drug permeation into and across the cell monolayer. Apical and basolateral media from a single culture were assayed in duplicate for parent drug and metabolites as described previously (Schmiedlin-Ren et al., 1997). For the analysis of intracellular MDZ, 1′-OH MDZ and 4-OH MDZ 0.02 to 0.8 ml of cell suspension were diluted with deionized water to a 1-ml volume before the solvent extraction step.

Determination of MDZ Permeability Coefficient.

The apparent permeability coefficient (Papp) of a drug across Caco-2 monolayers is often used as an in vitro predictor of in vivo drug absorption (Artursson, 1990; Gres et al., 1998). The Papp(cm/s) for MDZ in the modified Caco-2 monolayer was calculated according to the equation described by Artursson et al., (1990):Papp=dQdt·1AC0 Equation 1where dQ/dt is the rate of MDZ transfer (pmol/s) into the receiver compartment after an apical or basolateral MDZ dose under sink conditions (when <10% of the dose had crossed into the receiver compartment), A is the area of the culture insert (4.2 cm2), and C 0 is the concentration of MDZ in the dosing compartment at time zero. For these experiments, modified Caco-2 cells grown on laminin-coated inserts were treated apically or basolaterally with 3 μM MDZ.

Determination of First-Pass Metabolic Extraction Ratio (ER).

Initial experimental results allowed us to define linear, first-order conditions for metabolism during first-passage of MDZ across the Caco-2 cell monolayer. Briefly, MDZ was incubated under sink conditions (approximately 20 min in duration, when <10% of the apical dose had crossed into the basolateral compartment). The amounts of 1′-OH MDZ and 4-OH MDZ found in the apical, basolateral, and cell compartments were summed and entered into eq. 2, along with the amount of MDZ in the basolateral compartment, to calculate a first-pass ER:ERCaco−2Apical Dose= Equation 2 Σ(1′−OH MDZ)+Σ(4−OH MDZ)Σ(1′−OH MDZ)+Σ(4−OH MDZ)+MDZbasolateral When apical dosing concentrations of MDZ were relatively low (≤3 μM), it was found that 4-OH MDZ did not contribute significantly to total metabolite formation and it was not included in the calculated extraction ratios shown for interday variability.

Statistics.

A single enzyme, Michaelis-Menten model was fit separately to initial formation rates of 1′-OH and 4-OH MDZ at varying apical concentrations of MDZ using WinNonlin version 1.0 (Apex, NC) with a constant coefficient of variance error model. All other statistical analyses were performed with the statistical software SPSS version 7.5 (Chicago, IL). For dose-dependent metabolite kinetics, ANOVA was used to determine if there was a significant difference in mean parameter values for the four MDZ dose groups.

Results

TEER.

TEER (ohm · cm2) was recorded for all cells at 2 weeks postconfluence. Values ranged from 375 to 953 ohm · cm2 for 1α,25-(OH)2-D3-treated cells grown on hand-coated inserts and 315 to 688 ohm · cm2 for cells grown on commercially coated inserts. There was a statistically significant difference in mean TEER between untreated and 1α,25-(OH)2-D3-treated cells when grown on hand-coated (717 versus 589 ohm · cm2,p < .01) or commercially coated (1292 versus 471 ohm · cm2, p < .01) inserts. Additionally, we observed a decreasing TEER for confluent cells that were grown on hand-applied laminin-coated inserts and treated for an increasing number of days with 1α,25-(OH)2-D3(Fig. 1). A similar observation was recently reported by Chirayath et al., (1998) who described a 50% decrease in TEER in 2-week postconfluent Caco-2 cells treated with 10−8 M 1α,25-(OH)2-D3. Their results suggest that this decrease could be due to an increased permeability in the tight junctions between cells.

Figure 1
Figure 1

TEER measurements for Caco-2 cell monolayers grown on hand-applied laminin coated inserts and treated for increasing number of days with 1α,25-(OH)2-D3. Measurements for each 4.2 cm2 culture insert were obtained 14 days after the cells had reached confluence.

Stability of CYP3A4 Activity.

To make the cell culture system more amenable for studies of first-pass metabolism, FBS and 1α,25-(OH)2-D3 were removed from the apical (A) and basolateral (B) media during the MDZ incubation period. Because depletion of these agents would eventually alter cell CYP3A4 expression and metabolic activity, MDZ 1′-hydroxylation activity was evaluated after incubating cell monolayers for varying intervals over a 24-h period in culture medium lacking FBS and 1α,25-(OH)2-D3. As seen in Fig.2, the total amount of 1′-OH MDZ formed during a 30-min incubation with 3 μM apically applied MDZ was relatively stable for the first 4 h of serum-free incubation, after which time activity gradually declined.

Figure 2
Figure 2

Time-course of 1′-OH-MDZ formation activity in Caco-2 cell monolayers after conversion to serum-free, 1α,25-(OH)2-D3-free differentiation medium. Total (■), apical (□), basolateral (▴), and cellular (▵) 1′-OH MDZ content represent amounts measured after incubation for 30 min with apically-applied MDZ. Each point represents data obtained from a single culture.

Apical, basolateral, and intracellular 1′-OH MDZ all decreased in parallel with time. Twenty-four hours after removal of FBS and 1α,25-(OH)2-D3, the activity was found to be 27% of peak metabolite formation. Based on these initial findings, all incubation experiments with MDZ were limited to no more than 2 h in the absence of serum and 1α,25-(OH)2-D3.

Reproducibility of Cell Culture Activity.

A reliable assessment of first-pass kinetics depends on a reproducible measurement of the Caco-2 uptake and metabolism of MDZ during relatively short (≤30-min) incubation. Table 1 provides the intraday variability in 1′-OH MDZ appearance in the apical, basolateral, and cellular compartments from individual cultures after a 30-min incubation with 3 μM apical MDZ. Total 1′-OH MDZ formation was reasonably consistent across cultures (c.v. = 11.3%) as were apical/basolateral concentration ratios (c.v. = 8.2%). Cellular 1′-OH MDZ accounted for up to 18.7% of the total metabolite formed.

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Table 1

Intraday variability in 1′-OH MDZ formation and distribution in Caco-2 cells

MDZ Flux after Apical and Basolateral Administration.

Apical or basolateral dosing of MDZ (3 μM) allowed us to study the absorption (A → B) and exsorption (B → A) kinetics of this drug across the modified Caco-2 monolayer under nonsaturating conditions. Consistent with our previous findings (Schmiedlin-Ren et al., 1997), we observed a rapid decline in MDZ levels in both dosing compartments over the 2-h incubation period (Fig. 3a). Although the initial rate of loss of MDZ (0–60 min) from the basolateral compartment appeared slower than the loss from the apical compartment, the transcellular fluxes (as measured by the rate of MDZ accumulation in the receiving compartment) did not differ. Application of the initial rates of accumulation in the receiving compartments to eq. 1 yielded mean transcellular Papp values for MDZ dosed into either the apical (n = 5) or basolateral (n = 3) compartment of 28.5 ± 9.25 × 10−6 cm/s and 20.3 ± 4.56 × 10−6cm/s, respectively. There was no statistical difference in the Papp calculated after dosing into either compartment.

Figure 3
Figure 3

Time-course of incubation of 1α,25-(OH)2-D3-treated Caco-2 monolayers with MDZ. A 3 μM dose was applied to either the apical (open symbols) or basolateral (closed symbols) compartment. a, amount of MDZ in the apical (squares) or basolateral (triangles) compartments. b, transcellular MDZ concentration ratios. c, total 1′-OH MDZ (pmol) formation. Each point represents data obtained from a single culture. The dotted line in Fig. 3b indicates unity.

Consistent with a high level of permeability, MDZ transcellular (donor/recipient) concentration ratios declined sharply during the first 30 min of incubation after an apical or basolateral dose (Fig.3b). The ratio remained above unity at the end of the 2-h incubation interval, although the apical transcellular ratio for MDZ appeared to reach a plateau at a value just above unity after 1 h, whereas the basolateral ratio continued to decline towards that same level beyond 1 h. Our previous results (Schmiedlin-Ren et al., 1997) indicate that, after approximately 10 h of incubation with either an apical or basolateral dose, the MDZ concentrations will eventually equilibrate across the Caco-2 monolayer such that the apical concentration is 1.2- to 1.4-fold higher than the basolateral concentration.

Extracellular 1′-OH MDZ Partitioning.

We previously observed preferential sorting of 1′-OH MDZ into the apical compartment (compared to basolateral) after apical or basolateral dosing of MDZ in cultures incubated up to 24 h (Schmiedlin-Ren et al., 1997). As seen in Table 2, apically directed sorting of 1′-OH MDZ (∼2.4:1) also occurred in the absence of FBS and at shorter incubation intervals. In this experiment, apical, basolateral, and cellular 1′-OH MDZ were quantitated after 10 to 120 min of incubation with 3 μM MDZ dosed either apically or basolaterally. Although there was slightly more metabolite produced during the first 45 min after apical MDZ dosing, in comparison with basolateral dosing, total product formation was comparable during the 60- to 120-min interval (Fig. 3c). In addition, the apical/basolateral 1′-OH MDZ ratio was above unity for both routes of administration over the entire 2-h incubation period (Table 2).

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Table 2

Apical/basolateral 1′-OH MDZ concentration ratios

First-Pass MDZ Uptake and Metabolism.

Based on the apparent speed with which MDZ gained access to intracellular CYP3A, we examined the kinetics of MDZ metabolism during the first 10 min of incubation. After apical administration, MDZ diffused rapidly into the cell reaching a constant level within 4 to 6 min (Fig.4a). Cellular uptake of MDZ after a basolateral dose was much slower. It steadily increased over the 10-min incubation period, reaching an amount that was ∼ 13 of that achieved after apical dosing. Interestingly, addition of MDZ to both apical and basolateral compartments simultaneously caused only a 20% increase in the cellular MDZ level after 10 min of incubation over that observed after apical dosing alone.

Figure 4
Figure 4

Early time-course of incubation of 1α,25-(OH)2-D3-treated Caco-2 monolayers with MDZ. A 3 μM dose was applied to the apical (□), basolateral (▴), or both (■) compartments. a, MDZ uptake into Caco-2 monolayers. b, total 1′-OH MDZ (pmol) formation. c, transcellular flux of MDZ (A → B or B → A). Each point represents data obtained from a single culture incubated for the indicated time.

The cumulative amount of 1′-OH MDZ formed after each route of administration of MDZ corresponded to the amount of MDZ that accumulated in the cell monolayer (Fig. 4b). 1′-OH MDZ appeared immediately after apical MDZ administration and, after 10 min, was approximately 4-fold higher than the amount formed after basolateral dosing. Simultaneous administration of MDZ resulted in a 30% higher 1′-OH MDZ formation than after apical dosing, which was consistent with the modest increase in the cellular MDZ level (Fig. 4b).

To evaluate the possible presence of an apical or basolateral MDZ efflux pump, we examined MDZ transfer from the dosing compartment to the receiving compartment under sink conditions. Total 1′-OH MDZ formation at the end of a 10-min incubation was only 53 and 15 pmol, or 1.5% and 0.3% of the 4500 pmol (3 μM) apical and basolateral MDZ dose, respectively. As seen in Fig. 4c, the rates of transfer of MDZ from the apical to basolateral compartment (A → B) and from the basolateral to apical (B → A) compartment were linear during the 10-min incubation. Therefore, although the initial accumulation of MDZ in the cellular compartment was much lower after basolateral dosing (Fig. 4a), the rate of transfer of MDZ across the cell monolayer was roughly equal after apical and basolateral dosing (Fig. 4c).

Saturation Kinetics of Product Formation.

Under conditions of near constant intracellular MDZ concentrations (0–30 min after an apical dose), formation rates of 1′-OH and 4-OH MDZ were measured over a range of initial apical MDZ dosing concentrations (3.0–100 μM). Results are presented in Fig. 5 and Table 3. Each point represents the mean formation rate of three cultures incubated for 30 min with the indicated apical MDZ dose. The plot shows saturation of both the 1′-OH MDZ and 4-OH MDZ formation pathways in the Caco-2 cell monolayer at apical MDZ concentrations exceeding ∼25 and 80 μM, respectively. A single-enzyme, Michaelis-Menten kinetic model was fit to the data to yield apparent Michaelis constants (K m,app) of 9.1 and 84.7 μM and maximum formation rates (V max) of 11.1 and 6.1 pmol/min/culture for the 1′-OH and 4-OH MDZ formation pathways, respectively. Because the concentration of MDZ at the active site of the enzyme is unknown,K m,app is based on the initial apical MDZ concentration. However, the total cellular MDZ level correlated extremely well with the apical MDZ concentration after the 30-min incubation period (r = 0.998), indicating a rapid equilibration between the two compartments. The MDZ A/B concentration ratio increased slightly with an increase in the initial MDZ dose concentration; 3.74 ± 0.13, 4.24 ± 0.20, 4.63 ± 0.26, and 4.89 ± 0.68 for 3, 8, 25, and 100 μM MDZ. Mean values for the 3 and 100 μM dose groups were significantly different (p = .03).

Figure 5
Figure 5

Dependence of initial 1′-OH (●) and 4-OH MDZ (○) formation rates on MDZ concentration. Total 1′-OH and 4-OH MDZ formation were measured after a 30-min incubation with MDZ (3–100 μM) applied to the apical compartment. Each data point represents the mean rate of formation from three cultures. Error bars representing S.D. for some formation rates were too small to be seen on the plots. The dotted lines represent the fit of a single-enzyme, Michaelis-Menten model to the data; K m,app = 9.1 μM,V max = 11.1 pmol/min/culture (1′-OH MDZ);K m,app = 84.7 μM, V max= 6.1 pmol/min/culture (4-OH MDZ).

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Table 3

Dose-dependent metabolite kinetics and distribution in Caco-2 monolayers

Increasing apical dosing concentrations of MDZ also resulted in an increase in the contribution of 4-OH MDZ to total product formation (Fig. 5 and Table 3). Consistent with a trend seen previously in incubations with human duodenal microsomes (Paine et al., 1997), the ratio of 1′-OH to 4-OH MDZ in Caco-2 monolayers decreased from 12.3 to 3.1 as MDZ dosing concentrations increased from 3 to 100 μM (p < .001). At the lowest dosing concentrations, total 4-OH MDZ represented only 7.4% of total product formation. Similar to 1′-OH MDZ, 4-OH preferentially sorted (∼3.5:1) to the apical compartment after an apical dose, but neither metabolite showed any dose-dependent sorting over the indicated concentration range.

Determination of First-Pass MDZ ER.

Application of eq. 2 to the MDZ and 1′-OH MDZ results obtained from six separate experiments allowed us to determine an interday variability for the first-pass MDZ ER under subsaturating conditions (Table 4). ER values were calculated from single cultures incubated for 30 min or less with an apical MDZ dose concentration of 3 μM. The duration of MDZ incubation was selected based on the time when less than 10% of the apical MDZ dose had appeared in the basolateral compartment. Using these criteria, ER values between 11.4 and 19.0% were obtained, with a mean of 14.5 ± 3.1%.

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Table 4

Interday variability of ER in modified Caco-2 cells

A first-pass ER was also calculated for the Caco-2 monolayers dosed apically with increasing concentrations of MDZ (3–100 μM) using 1′-OH and 4-OH MDZ measurements (Table 3). Although the incubation period was limited to 30 min, the percentage of the initial MDZ dose that appeared in the basolateral compartment exceeded our preset limit of 10% (13.1 to 18.1%). Under this condition, increasing dosing concentrations resulted in a significant reduction in the apparent first-pass ER from 9.8 ± 1.2% (3 μM) to 1.6 ± 0.26% (100 μM; p < .001).

Immunoquantitation of CYP3A in Modified Caco-2 Cells.

Total CYP3A content in modified Caco-2 cells was determined by Western blot analysis of a pooled homogenate prepared from 28 replicate monolayer cultures. CYP3A4 appeared to be the only protein in 1α,25-(OH)2-D3-treated cells detected by the anti-CYP3A antibody (Fig. 6). Quantitation of the homogenate CYP3A4 band density relative to standard indicated a specific content of 3.7 pmol/4.2 cm2 cultured insert. Analysis of the 1st centrifugation pellet (21,400g) prepared from homogenate indicated that approximately 90% of CYP3A protein was removed with the plasma membrane and mitochondrial fragments. Consequently, total CYP3A yield in the 2nd pellet (110,000g) was only 5% of the original amount. The specific content for 1α,25-(OH)2-D3 treated Caco-2 cell microsomes was found to be 8.3 pmol/mg protein.

Figure 6
Figure 6

Western blot of 1α,25-(OH)2-D3-treated Caco-2 cell fractions and microsomes from human liver and small intestine. Caco-2 cell homogenate (50 μg), 1st centrifugation 21,400g pellet (50 μg), 1st 21,400g supernatant (40 μg), and final 110,000g centrifugation pellet (20 μg) and human intestinal (20 μg) and liver (10 μg) microsomes were electrophoresed in parallel to expressed CYP3A4 and CYP3A5 standards (0.5 and 1 pmol each). Resolved proteins were transferred to nitrocellulose and probed with an anti-human CYP3A4 polyclonal antibody, as described in Experimental Procedures.

Discussion

Although Caco-2 cells are an excellent model for the prediction of drug absorption across the intestinal epithelium, until recently they have not been used as an in vitro model for P-450-mediated drug metabolism. When treated with 1α,25-(OH)2-D3for 2 weeks postconfluence, CYP3A4 mRNA and protein, the dominant P-450 isoform in human intestinal mucosa (de Waziers et al., 1990; Paine et al., 1997), were increased substantially in the Caco-2 cell monolayer (Schmiedlin-Ren et al., 1997). Results from the present study show that CYP3A-induced Caco-2 monolayers can also be used as a model for first-pass intestinal drug metabolism.

MDZ is a short-acting, water-soluble benzodiazepine that is rapidly and nearly completely absorbed after oral administration (T max 15–30 min; Heizmann and Ziegler, 1981;Smith et al., 1981). Consistent with these physicochemical properties, we observed a rapid uptake of MDZ into the cell monolayer and a rapid flux of MDZ from the apical to basolateral compartment, as described by a high Papp of 28.5 × 10−6 cm/s. Many substrates for CYP3A4 are transported across barrier cell plasma membranes by P-gp (Wacher et al., 1995). For example, efficient P-gp-mediated luminally-directed efflux of cyclosporine from intestinal epithelial cells appears to delay and limit the oral bioavailability of the drug (Lown et al., 1997). For cyclosporine and other P-gp substrates, transcellular flux in Caco-2 monolayers is faster in the (B → A) direction than in the (A → B) direction. Thus, the nearly equivalent rates of MDZ accumulation in the receiving compartment we observed after apical or basolateral MDZ administration (Fig. 4c) suggest that MDZ is not a significant substrate for the P-gp efflux transporter. One caveat to this conclusion is the modest 31% increase in the 30-min A/B MDZ concentration ratio that was observed with increasing (3- to 100-μM) MDZ dose concentration. However, this trend was more consistent with the saturation of a basolaterally directed active transport process and not an apically directed transporter such as P-gp. In addition, our previous results from Caco-2 monolayer incubation with MDZ in the presence of the selective P-gp inhibitor verapamil (Schmiedlin-Ren et al., 1997) also support this conclusion.

A plausible explanation of the delayed rate of MDZ uptake into the cell monolayer from the basolateral compartment (Fig. 4a), in comparison with the apical compartment, is the difference between apical and basolateral surface area and the presence of the laminin barrier. The large surface area of the apical membrane promoted a rapid uptake of MDZ into the cell mass after apical administration, whereas the smaller surface area and presence of the laminin coating resulted in a relatively slow uptake from the basolateral compartment over a 10-min incubation period. Identification of the rate-limiting step for the delayed uptake of MDZ from the basolateral compartment was beyond the scope of this study.

In principle, the rate of cellular uptake of a CYP3A4 substrate may have a significant effect on the extent of first-pass and systemic intestinal metabolism. We examined this by administration of MDZ to one or both extracellular compartments. When the rate of diffusional efflux (apical or basolateral dose) from the intracellular compartment was rendered negligible by high MDZ concentrations in the receiver compartment (Fig. 4, a and b), one might have expected greater cellular MDZ accumulation and product formation than when an extracellular sink was present. However, the absence of such findings suggests that after an apical (oral) MDZ dose, the initial pseudo-steady-state intracellular MDZ concentration is controlled primarily by the apical MDZ concentration and the intracellular metabolic clearance, and not MDZ back-diffusion across the basolateral membrane.

After its formation, 1′-OH MDZ movement out of the modified Caco-2 cell monolayer could be governed by diffusion or active efflux transport. In both this study and our previous work (Schmiedlin-Ren et al., 1997), 1′-OH MDZ preferentially sorted to the apical compartment (A/B ratios greater than unity at all incubation times) regardless of the administration compartment. Similar results were found in the presence of FBS in both compartments after co-administration of 100 μM verapamil (Schmiedlin-Ren et al., 1997), suggesting that the 1′-OH MDZ sorting mechanism is also not controlled by the apically directed P-gp pump that is expressed in these cells. This does not rule out the possibility of an alternate transport mechanism. However, the apparent preference for metabolite distribution into the apical compartment could also be explained by a purely diffusional mechanism. Under conditions where the enzyme provides a continuous generation of 1′-OH MDZ in the cellular (C) compartment, it might be expected that the greater surface area of the apical plasma membrane, compared with the basolateral membrane, would result in a C → A flux of metabolite that exceeds the C → B flux. A true equilibrium would not be achieved unless MDZ metabolism were to cease. Further studies are needed to discriminate between an active transport system and simple diffusion. However, under more physiologically relevant conditions, with extensive (∼90%) plasma protein binding of the MDZ metabolite (Mandema et al., 1992), blood in the capillaries of the lamina propria may act as a sink and override any “preferred” apical (luminal) 1′-OH MDZ sorting.

Saturable enzyme kinetics were also evaluated in the modified cell monolayer by measuring total 1′-OH and 4-OH MDZ formation as a function of increasing concentrations of apically applied MDZ. A single-enzyme, Michaelis-Menten model appeared to describe both metabolite formation rate versus initial apical MDZ concentration plots (Fig. 5). There was no evidence of enzyme inactivation or nonhyperbolic kinetics at high concentrations of MDZ, although an extensive profiling of the concentration-velocity curve was not performed. The estimatedK m,app of 84.7 μM for 4-OH MDZ in Caco-2 cultures was similar to (Gorski et al., 1994) or higher than (Ghosal et al., 1996) the mean value reported with human liver microsomes (86.5 μM) and cDNA expressed CYP3A4 (38 μM), respectively. The estimatedK m,app of 9.1 μM for 1′-OH MDZ in the Caco-2 monolayer was also higher than the median K mvalue reported for microsomes from human small intestine (3.8, 3.7, and 4.5 μM for duodenum, jejunum, and ileum, respectively; Paine et al., 1997) and the K m for cDNA-expressed CYP3A4 (1.6 μM; Ghosal et al., 1996). These data suggest that under pseudo-steady-state conditions, the mean unbound intracellular concentration of MDZ at the CYP3A4 active site is less than one-half the apical MDZ concentration. Diffusion of MDZ out of the cell monolayer and a high rate of metabolism could potentially have reduced the unbound intracellular concentration at the enzyme active site relative to the apical concentration, yielding a higher calculatedK m,app. Relative rates of cell uptake from the dosing compartment, intracellular metabolism and diffusion into the receiver compartment may be similarly important factors to consider when evaluating the metabolic extraction of other CYP3A4 substrates.

Based on the reproducibility of the rate of metabolite formation of 1′-OH MDZ and its short-term stability in the absence of FBS and 1α,25-(OH)2-D3, the modified Caco-2 monolayer appears well suited for the study of first-pass metabolism kinetics. Although it is possible to adapt cell culture medium with the proper nutrients and growth factors such that cells can be cultured in the absence of serum, it is generally believed that serum is essential to both membrane stability and cell function. The stability of the 1′-OH MDZ formation rate is likely to reflect the stability of the CYP3A enzyme level in the modified Caco-2 monolayer. We previously estimated by immunoquantification (Schmiedlin-Ren et al., 1997) that the 1α,25-(OH)2-D3-treated monolayer contains 20.6 pmol CYP3A/mg homogenate protein. This translates into an estimated 25 pmol of CYP3A per 4.2 cm2 cultured insert. However, our current Western blot result suggests the CYP3A content is much lower than initially estimated (3.7 pmol/4.2 cm2cultured insert). One reason for this disparity in CYP3A4 content may be the different Western blot development techniques and standards used for the two studies. Despite this apparent difference in CYP3A content, the initial (0–2 h) 1′-OH MDZ formation rate was comparable after apical MDZ incubations in both studies, suggesting that active CYP3A4 protein content was similar and stable in both systems.

We can also approximate CYP3A4 content in our modified Caco-2 cell monolayer on the basis of the expected and observed turnover number for MDZ 1′-hydroxylation. Turnover numbers of 1.6 pmol/pmol CYP3A4/min (Gorski et al., 1994) and 5.6 pmol/pmol CYP3A4/min (Gibbs et al., 1999) have been reported for purified and cDNA-expressed enzymes, respectively. Based on these values and our measuredV max value with Caco-2 cells, the expected content of CYP3A in the cell monolayer would be 6.9 and 2.0 pmol per culture, respectively, which are in close agreement with the immunoblot results observed in this study (3.7 pmol/culture).

The mean first-pass ER obtained from the cell cultures, 14.5 ± 3.1%, is at the low end of a range of values (13.6–58.6%) obtained from an in vivo study where intestinal MDZ extraction to 1′-OH MDZ was measured directly in liver transplant patients (Paine et al., 1996). One reason for an apparent in vitro—in vivo discrepancy may be a lower level of expression of active CYP3A4 enzyme in the Caco-2 system, compared with intestinal enterocytes (8.3 versus 30.6 pmol/mg microsomal protein for Caco-2 cells from the present study and human duodenal mucosa, respectively; Paine et al., 1997), as discussed above. Although total CYP3A content in homogenates prepared from the Caco-2 monolayer and intestinal mucosal scrapings were relatively similar (7.7 versus 9.2 pmol/mg homogenate protein), total homogenate protein from the human intestine includes proteins from luminal contents and villous cells other than enterocytes whereas Caco-2 homogenate protein is from a single cell type. This difference in homogenate protein composition could yield a lower CYP3A content in human mucosal homogenate preparations relative to preparations from enterocytes alone. Thus, the somewhat low first-pass MDZ extraction efficiency of the 1α,25-(OH)2-D3-modified Caco-2 monolayer, compared with the in vivo ER, may actually be in line with the observed difference in CYP3A expression. If this proves to be true, scaling factors may be developed to increase the in vitro to in vivo predictive power of the Caco-2 system.

In summary, our results describe for the first time the use of a Caco-2 cell monolayer as an in vitro model to simulate intestinal first-pass metabolic extraction kinetics for a CYP3A-catalyzed reaction. Using MDZ 1-hydroxylation as an enzyme-selective probe, we obtained an average first-pass extraction ratio of ∼15% for a subsaturating apically applied MDZ dose. This level of metabolic activity is clearly sufficient to support investigations of the effect of extracellular protein (i.e., basolateral plasma proteins) as well as the effects of enzyme modulators (i.e., inhibitors and activators) on the first-pass elimination process. The model system should also be suitable for examination of the first-pass metabolic extraction of other moderate to high intrinsic clearance CYP3A substrates. Further, because the 1α,25-(OH)2-D3-treated Caco-2 cells also express P-gp (Schmiedlin-Ren et al., 1997), they should also be appropriate for studies investigating the dynamic interplay between metabolism, active efflux, and transcellular permeability.

Footnotes

  • Send reprint requests to: Dr. Kenneth E. Thummel, Ph.D., Department of Pharmaceutics, Box 357610, University of Washington, Seattle, WA 98195-7610. E-mail: thummel{at}u.washington.edu

  • ↵1 This study was supported in part by Eli Lilly & Co. and National Institutes of Health Grant GM 32165.

  • Abbreviations:
    CYP
    cytochrome P-450
    P-gp
    P-glycoprotein
    MDZ
    midazolam
    1′-OH MDZ
    1′-hydroxymidazolam
    1α
    25-(OH)2-D3, 1α,25-di-hydroxy vitamin-D3
    FBS
    fetal bovine serum
    DMEM
    Dulbelcco’s modified Eagle’s medium
    NEAA
    non-essential amino acids
    DM
    differentiation medium
    PET
    polyethylene terephthalate
    TEER
    transepithelial electrical resistance
    DMSO
    dimethylsulfoxide
    ER
    extraction ratio
    • Received October 19, 1998.
    • Accepted January 16, 1999.

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First-Pass Midazolam Metabolism Catalyzed by 1α,25-Dihydroxy Vitamin D3-Modified Caco-2 Cell Monolayers

Jeannine M. Fisher, Steven A. Wrighton, Paul B. Watkins, Phyllissa Schmiedlin-Ren, Justina C. Calamia, Danny D. Shen, Kent L. Kunze and Kenneth E. Thummel
Journal of Pharmacology and Experimental Therapeutics May 1, 1999, 289 (2) 1134-1142;
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